The present invention relates generally to the biosynthesis of glycans found as free oligosaccharides or covalently bound to proteins and glycosphingolipids. This invention is more particularly related to a family of nucleic acids encoding UDP-D-galactose: xcex2-N-acetylglucosamine xcex2-1,4-galactosyltransferases (xcex24Gal-transferases), which add galactose to the hydroxy group at carbon 4 of 2-acetamido-2-deoxy-D-glucose (GlcNAc). This invention is more particularly related to a gene encoding the second member of the family of xcex24Gal-transferases, termed xcex24Gal-T2, probes to the DNA encoding xcex24Gal-T2, DNA constructs comprising DNA encoding xcex24Gal-T2, recombinant plasmids and recombinant methods for producing xcex24Gal-T2, recombinant methods for stably transfecting cells for expression of xcex24Gal-T2, and methods for identification of DNA polymorphism in patients.
The UDP-galactose: xcex2-N-acetyl-glucosamine xcex2-1,4-galactosyltransferase (xcex24Gal-T1) was the first animal glycosyltransferase to be isolated and cloned (Narimatsu et al., 1986; Shaper et al., 1986; Nakazawa et al, 1988; Shaper et al., 1988; D""Agostaro et al., 1989), and early searches for homologous genes by low stringency Southern hybridisation suggested that this gene was unique. Characterisation of xcex24Gal-transferase activities from different sources, however, indicate that distinct activities exist (Sheares and Carlson, 1984; Furukawa et al., 1990). Emerging evidence now reveal that several xcex24galactosyltransferase genes may exist. Shaper and colleagues (Shaper et al., 1995) have identified two different chick cDNA sequences, which have 65% and 48% sequence similarity to human xcex24Gal-T1. Both chick cDNAs were shown to encode catalytically active p4Gal-transferases (Shaper et al., 1997). Two independent groups have analysed xcex24Gal-transferase activities in mice homozygously deficient for xcex24Gal-T1 (Asano et al., 1997; Lu et al., 1997). Both studies showed residual xcex24Gal-transferase activity, providing clear evidence for the existence of additional xcex24Gal-transferases. Thus, the xcex24Gal-T1 gene is likely to be part of a homologous gene family with recognisable sequence motifs, and this is supported by a large number of human ESTs with sequence similarities to xcex24Gal-T1 in EST databases (National Center for Biotechnology Information).
xcex2-1,4-Galactosyltransferase activities add galactose to different acceptor substrates including free oligosaccharides, N- and O-linked glycoproteins, and glycosphingolipids (Kobata, 1992). In addition, xcex24Gal-T1 is modulated by xcex1-lactalbumin to function as lactose synthase and hence has a major role in lactation (Brew et al., 1968). Given the diverse functions of xcex2-1,4-galactosyltransferase activities and the evidence that multiple xcex24Gal-transferases exist, it is likely that these enzymes may have different kinetic properties. Furukawa et al (Furukawa et al., 1990) showed that liver xcex24Gal-transferase activity was near 20-fold higher with asialo-agalacto-transferrin compared to asialo-agalacto-IgG, whereas the activity found in T and B cells only showed a 4 to 5-fold difference with the two substrates. The xcex24Gal-transferase activity in B cells of rheumatoid arthritis patients appear to be similar to B cells from healthy controls with several substrates including asialo-agalacto-transferrin (Furukawa et al., 1990) and xcex2GlcNAc-pITC-BSA (Keusch et al., 1995), but different with asialo-agalacto-IgG (Furukawa et al., 1990). Furthermore, the Km for UDP-Gal of xcex24Gal-transferase activity from B cells of rheumatoid arthritis patients were 2-fold higher (35.6 mM) than normal B cells (17.6 mM) (Furukawa et al., 1990). Finally, the activity in B cells for asialo-agalacto-transferrin was more sensitive to xcex1-lactalbumin inhibition than the activity with asialo-agalacto-IgG. A number of studies have concluded that there was no change in xcex24Gal-transferase activity in B cells of rheumatoid arthritis patients (Wilson et al., 1993; Axford et al., 1994). However, if multiple xcex24Gal-transferases exist, it is possible that the contradictory findings of Furukawa et al. (Furukawa et al., 1990) can be explained by a model with two xcex24Gal-transferases with different kinetic parameters expressed in normal B cells, and a selective down regulation of one in B cells of rheumatoid arthritis patients.
Access to additional existing xcex24Gal-transferase genes encoding xcex24Gal-transferases with better kinetic properties than xcex24Gal-T1 would allow production of more efficient enzymes for use in galactosylation of oligosaccharides, glycoproteins, and glycosphingolipids. Such enzymes could be used, for example, in pharmaceutical or other commercial applications that require synthetic galactosylation of these or other substrates that are not or poorly acted upon by xcex24Gal-T1, in order to produce appropriately glycosylated glycoconjugates having particular enzymatic, immunogenic, or other biological and/or physical properties.
Consequently, there exists a need in the art for additional UDP-galactose: xcex2-N-acetyl-glucosamine xcex2-1,4-galactosyltransferases and the primary structure of the genes encoding these enzymes. The present invention meets this need, and further presents other related advantages.
The present invention provides isolated nucleic acids encoding human UDP-galactose: xcex2-N-ace-tylglucosamine xcex21,4-galactosyltransferase (xcex24Gal-T2), including cDNA and genomic DNA. xcex24Gal-T2 has better kinetic parameters than xcex24Gal-T1, as exemplified by its lower Km for UDP-Gal and its better activity with saccharide derivatives, glycoprotein substrates, and xcex2GlcNAc-glycopeptides. The complete nucleotide sequence of xcex24Gal-T2, SEQ ID NO:1, is set forth in FIG. 2.
In one aspect, the invention encompasses isolated nucleic acids comprising the nucleotide sequence of nucleotides 1-1116 as set forth in SEQ ID NO:1 or sequence-conservative or function-conservative variants thereof Also provided are isolated nucleic acids hybridizable with nucleic acids having the sequence of SEQ ID NO:1 or fragments thereof or sequence-conservative or function-conservative variants thereof; preferably, the nucleic acids are hybridizable with xcex24Gal-T2 sequences under conditions of intermediate stringency, and, most preferably, under conditions of high stringency. In one embodiment, the DNA sequence encodes the amino acid sequence, SEQ ID NO:2, also shown in FIG. 2, from methionine (amino acid no. 1) to glycine (amino acid no. 372). In another embodiment, the DNA sequence encodes an amino acid sequence comprising a sequence from tyrosine (no. 31) to glycine (no. 372) of SEQ ID NO:3.
In a related aspect, the invention provides nucleic acid vectors comprising xcex24Gal-T2 DNA sequences, including but not limited to those vectors in which the xcex24Gal-T2 DNA sequence is operably linked to a transcriptional regulatory element, with or without a polyadenylation sequence. Cells comprising these vectors are also provided, including without limitation transiently and stably expressing cells. Viruses, including bacteriophages, comprising xcex24Gal-T2-derived DNA sequences are also provided. The invention also encompasses methods for producing xcex24Gal-T2 polypeptides. Cell-based methods include without limitation those comprising: introducing into a host cell an isolated DNA molecule encoding xcex24Gal-T2, or a DNA construct comprising a DNA sequence encoding xcex24Gal-T2; growing the host cell under conditions suitable for xcex24Gal-T2 expression; and isolating xcex24Gal-T2 produced by the host cell. A method for generating a host cell with de novo stable expression of xcex24Gal-T2 comprises: introducing into a host cell an isolated DNA molecule encoding xcex24Gal-T2 or an enzymatically active fragment thereof (such as, for example, a polypeptide comprising amino acids 31-372 of SEQ ID NO:2), or a DNA construct comprising a DNA sequence encoding xcex24Gal-T2 or an enzymatically active fragment thereof, selecting and growing host cells in an appropriate medium; and identifying stably transfected cells expressing xcex24Gal-T2. The stably transfected cells may be used for the production of xcex24Gal-T2 enzyme for use as a catalyst and for recombinant production of peptides or proteins with appropriate galactosylation. For example, eukaryotic cells, whether normal or diseased cells, having their glycosylation pattern modified by stable transfection as above, or components of such cells, may be used to deliver specific glycoforms of glycopeptides and glycoproteins, such as, for example, as inmunogens for vaccination.
In yet another aspect, the invention provides isolated xcex24Gal-T2 polypeptides, including without limitation polypeptides having the sequence set forth in SEQ ID NO:2, polypeptides having the sequence of amino acids 31-372 as set forth in SEQ ID NO:3, and a fusion polypeptide consisting of at least amino acids 31-372 as set forth in SEQ ID NO:3 fused in frame to a second sequence, which may be any sequence that is compatible with retention of xcex24Gal-T2 enzymatic activity in the fusion polypeptide. Suitable second sequences include without limitation those comprising an affinity ligand or a reactive group.
In another aspect of the present invention, methods are disclosed for screening for mutations in the coding region (exons I-VII) of the xcex24Gal-T2 gene using genomic DNA isolated from, e.g., blood cells of patients. In one embodiment, the method comprises: isolation of DNA from a patient, PCR amplification of coding exons I-VII; DNA sequencing of amplified exon DNA fragments and establishing therefrom potential structural defects of the xcex24Gal-T2gene associated with disease.